Microarray analysis of E2Fa-DPa-overexpressing plants uncovers a cross-talking genetic network between DNA replication and nitrogen assimilation

Abstract

IntroductionProgression through the cell cycle is essential for the continued existence of all uni-and multicellular organisms. It is crucial for the survival of a cell that its DNA is correctly replicated. In mammals, the onset of DNA replication is regulated by the activity of the heterodimeric E2F-DP transcription factor. The mammalian E2F family contains six proteins (E2F1, E2F2, E2F3, E2F4, E2F5 and E2F6) (Trimarchi and Lees, 2002). All E2Fs have an N-terminally located DNA-binding domain immediately followed by a dimerization domain, allowing them to pair with a dimerization partner (DP1 or DP2). Dimerization of E2F with DP is a prerequisite for high affinity, sequence-specific binding to the E2F consensus DNA-binding site. E2F activity is negatively regulated by retinoblastoma (Rb), which binds to the transcriptional activation domain of the E2F-DP factor, rendering it inactive. Moreover, the recruitment by Rb of DNA-modifying enzymes, such as histone deacetylases and polycomb proteins, leads to chromatin condensation with suppression of promoter activity of E2F-DP target genes as a result. Phosphorylation of Rb by cyclin-dependent kinases (CDKs) counteracts its inhibitory function, resulting in the release of transcriptionally active E2F-DP and consequential onset of DNA replication.The mechanism of DNA replication seems to be conserved between mammals and plants, because E2F and DP genes have been isolated from different plant species, including wheat,

Divide and conquer: development and cell cycle genes in plant transformation

“…To aid recognition of meristemoids at early stages in development, the use of transgenic marker genotypes, such as those containing a WUSCHEL-reporter or STM-reporter construct [58,59], would be useful. In addition, monitoring of S-phase-specific genes, such as RNR, MCM, CDC6, PCNA, H2A, H4 and E2Fa [23][24][25]38], might give detailed insights into the mechanisms of cell cycle modulation: using microarray analysis, O100 genes related to S phase have been identified in Arabidopsis after overexpression of E2Fa-DPa [24]. In addition, changes in heterochromatin remodeling, DNA or histone methylation and histone acetylation and deacetylation can be directly monitored by a variety of methods [17][18][19]60].…”

“…This phenotype was accompanied by a significantly higher proportion of 2C and 4C nuclei and two additional endocycles compared with the control [27]. Transcriptome analysis of E2Fa-DPa-overexpressing plants showed the up-regulation of 14 genes that are involved in DNA replication (and/or modification) and S phase onset, including histone acetylase B (HAT B), topoisomerase 6, DNA gyrase, CDKB1, and histones H1, H2A, H2B and H4 [24]. However, not all E2F factors positively regulate the G1-S transition.…”

“…Upon phosphorylation by D-and A-type cyclins, in association with their cyclin kinase partners, RBR releases the E2F factors, enabling the G1-S transition to occur [20,21,23]. The transcription activation factor E2Fa acts in combination with its dimerization partner DPa, and among the hundreds of genes targeted by E2Fa-DPa [24] are D-type cyclins (CycD), which provide feedback to the release of E2F factors in the E2F-RBR pathway [20,21]. It has been shown that Cyclin D3 (CycD3) expression is modulated in plant cells by the hormone cytokinin [25].…”

“…A H2A-like protein was also one of the genes up-regulated by the co-expression of E2Fa-DPa and S phase onset [24]. A mutation in H2A-1 rendered plants resistant to genetic transformation with Agrobacterium, whereas its overexpression in wild-type plants increased transformation efficiency by twofold [32].…”

E2F RegulatesFASCIATA1, a Chromatin Assembly Gene Whose Loss Switches on the Endocycle and Activates Gene Expression by Changing the Epigenetic Status

“…This suggests a complex regulatory network in which different combinations of E2F may affect FAS1 expression, possibly in a cell type-dependent manner. This idea of complexity in E2F regulation has a precedent in the data obtained in mRNA-profiling experiments of plants with increased levels of E2Fa, considered a typical activator (Vlieghe et al, 2003;Vandepoele et al, 2005). These studies identified genes whose expression was activated as well as genes whose expression was repressed, although, in both cases, some of them contain E2F binding sites in their promoters.…”